DLL4-Notch signaling is important for expression of pericyte markers in vitro. (A) Whole BM was isolated from the femurs of transgenic mice and grown in coculture with SC9-19 mouse embryonic stromal cells for 1 week. GFP⁺ BM cells were then isolated from the cocultures by FACS, and RNA was extracted. RT-PCR was performed for desmin and RGS5 on freshly isolated whole BM and on BM cells after 1 week in coculture. (B-C) GFP⁺ BM cells were isolated from the femurs of transgenic mice and grown in coculture with SC9-19 mouse embryonic stromal cells. Cells were treated daily with either IgG or YW152F (B) or dimethylsulfoxide or DAPT (C) for 1 week. GFP⁺ BM cells were then isolated from the cocultures by FACS and RNA was extracted. RT-PCR was performed to quantify RGS5, desmin, α-SMA, or Hes1 mRNA levels. Densitometry values are presented below each band. Experiments were repeated at least 2 additional times with similar results; data shown are representative. (D) 10T1/2 mesenchymal precursor cells were transfected with either MigR1 vector control or MigR1 containing the active domains of each of the 4 Notch receptors (NICD1-4). Forty-eight hours later, RNA was collected and mRNA levels were measured by RT-PCR for RGS5, α-SMA, and desmin. Experiments were repeated 3 times with similar results; results are representative. (E) TC71 cells were injected subcutaneously into nude mice that had previously received GFP⁺ BM transplants. When tumors reached 2 cm³, mice were killed and tumors were harvested and digested to form single-cell suspensions. GFP⁺ BM-derived cells were collected using FACS, and RNA was isolated from these tumor-derived GFP⁺ BM cells. RT-PCR was performed for DLL4 and Hes1.